Antibody can influence the fate of bacteria in vivo by lysis or opsonisation. The former mechanism is of unknown significance. Opsonins, however, are important whenever the parasite can resist ingestion but is susceptible to inactivation within the phagocytes. Some organisms, however, can multiply within cells. The antibody response to such organisms is often at variance with the degree of resistance developed against them (1, 2). This has been taken as evidence for the existence of an alternative, non-humoral form of acquired resistance. It is widely held that the alternative mechanism is mediated by cells; but this view has not been wholly acceptable (2, 3), and the immunological basis for it has never been explained. Tuberculosis and brucellosis are two infections in which a cellular component has been claimed to play a role in acquired resistance, but they are ill-suited to an experimental approach to the problem. For one thing the immune state is not particularly efficient in either of them. But this is perhaps of less importance than the technical problems encountered in working with the tubercle bacillus, and the fact that virulent strains of Brucella are susceptible both to opsonisation (4) and to lysis by complement in the presence of specific antibody (5). Unless antibody can be excluded as a factor in acquired resistance, difficulties arise in attempting to assign significance to non-humoral factors even though they may represent the major component of the host's defense mechanism.A series of studies has been undertaken to analyse the mechanism of acquired resistance to a facultative intracellular parasite, and the immunological processes upon which it is based. It was realised at the outset that this would be possible only if the combination of host and parasite was chosen so that technical problems were minimised, and the immune state were efficient enough to make its demonstration possible in vitro.The present series of investigations deal with the pathogenesis of listeriosis in mice. The course of the investigation was dictated by several considerations: that non-humoral immunity could express itself in a pure form only against a true facultative intracellular parasite; that the mouse develops a true acquired resistance to Lisleria (6); and that resistance to Lisleria monocytogenes is not 381 on

It has been shown that the immune response of mice to infection with L. monocytogenes gives rise to a population of immunologically committed lymphoid cells which have the capacity to confer protection and a proportionate level of delayed-type hypersensitivity upon normal recipients. The cells were most numerous in the spleen on the 6th or 7th day of infection, but persisted for at least 20 days. Further study revealed that the immune cells must be alive in order to confer protection, and free to multiply in the tissues of the recipient if they are to provide maximum resistance to a challenge infection. The antibacterial resistance conferred with immune lymphoid cells is not due to antibacterial antibody; it is mediated indirectly through the macrophages of the recipient. These become activated by a process which appears to depend upon some form of specific interaction between the immune lymphoid cells and the infecting organism. This was deduced from the finding that immune lymphoid cells from BCG-immunized donors, which were highly but nonspecifically resistant to Listeria, failed to protect normal recipients against a Listeria challenge unless the recipients were also injected with an eliciting dose of BCG. The peritoneal macrophages of animals so treated developed the morphology and microbicidal features of activated macrophages. It is inferred that acquired resistance depends upon the activation of host macrophages through a product resulting from specific interaction between sensitized lymphoid cells and the organism or or its antigenic products. Discussion is also made of the possibility that activation of macrophages could be dependent upon antigenic stimulation of macrophages sensitized by a cytophilic antibody.

Infection with certain organisms is often found to produce a high degree of resistance towards other, unrelated organisms (1). The phenomenon is conspicuous whenever the immunising infection is caused b y a facultafive intracellular parasite (2). The latter provokes an immune response with three peculiarities: a consistent association with delayed-type hypersensitivity, a form of acquired resistance that cannot be passively transferred with serum, and a reported change in the antibacterial activity of the host's macrophages.I t is usual to dismiss the cross-resistance caused b y such organisms as an example of "non-specific immunity," and to ignore the possible immunological significance of the phenomenon. I t was decided to reinvestigate the problem in the hope of arriving at a better understanding of the immunological processes involved in this type of acquired antibacterial resistance. In this report the specificity of both the induction and the expression of acquired resistance has been studied in mice immunised with three facultative intracellular parasites;Listeria monocytogenes, Brucella abortus, and Mycobacterium tuberculosis. Materials and MethodsMice.--The mice used were of an outbred strain (3). Animals of the one sex and of comparable age (7 to 8 weeks) were used in individual experiments.Organisms.PThe strain of L. monocytogenes (NCTC 7973, serotype I) was sustained at high virulence by continuous passage in normal mice. The attenuated vaccine strain of Br. abort, us (strain 19) was obtained from the Commonwealth Serum Laboratories (Victoria). Both organisms were grown in brain-heart infusion broth (BI-II, Difco Laboratories, Inc., Detroit). Suspensions of bacteria were prepared from 16-hour and 3-day cultures of L. monocytogenes and Br. abortus, respectively. The organisms were washed once and resuspended in Hanks' solution containing 0.1 per cent bovine serum albumin (fraction V, Armour). The LDs0 for L. monocy-/ogenes was 1.0 X 105 by intravenous injection; that for Br. abor/us (strain 19) was not determined.Bacterial Enumeration in the Spleen.--The method of determining the viable bacterial count in the spleen was previously described (3). Colony counts were made after 24 hours and 4 days for/./stcr/a and Brucdla cultures respectively.Dete~$ion of Listcria-Resistant Macrophages.--The method used for preparing and parasifis-

If it were possible to promote or suspend the formation of specifically sensitized lymphocytes (activated T cells), the problems of achieving a sustained attack on tumor cells and microbial parasites, or of preventing graft rejection, might be largely overcome. Methods for manipulating the immune response for such purposes have been proposed from time to time (1-3), but progress has been slow because so little is known of how cell-mediated immunity is normally regulated. Although much has been learned about the allied problem cf what controls the formation of antibodies (4), almost nothing is known about the mechanism that regulates the production and function of the cells which mediate delayed-type hypersensitivity (DTH).I While studying the tumor-suppressive activity of Mycobacterium boris BCG it was observed that lymphoid tissues which were under the stimtflatory influence of a BCG infection were capable of a much more vigorous response to a second antigen (5). Both cellular and humoral immunity to sheep red blood cells (SRBC) were augmented, as evidenced by higher and more sustained levels (DTH) and increased numbers of plaque-forming cells (PFC) in responding lymph nodes. Since D T H does not usually appear unless special conditions of immunization are used, these findings suggested that the formation of activated T cells is normally restricted by an inhibitory mechanism that does not operate properly in lymphoid tissues infected with BCG.Miller et al. (5) have shown that mice given a subcutaneous injection of SRBC in saline develop a poorly sustained state of hypersensitivity which conforms to all of the established criteria by which D T H is recognized, including its mediation by 0-bearing lymphocytes. It was therefore possible to study the mechanism which regulates T-cell activity in the absence of any influence from adjuvants such as were used by Nelson and Mildenhall (6) when they, too, showed that mice develop classical D T H in response to SRBC.

Experiments in vitro comparing normal mouse peritoneal macrophages with cells from Salmonella typhimurium-infected mice have shown that the "immune" macrophages have conspicuously enhanced microbicidal properties. Whereas normal macrophages could inactivate only 50 to 60% of intracellular S. typhimurium pretreated with immune serum, cells from infected animals killed virtually all ingested organisms and did so at an accelerated rate. Macrophages from Listeria monocytogenes-infected mice were shown to possess similarly enhanced microbicidal activity against S. typhimurium. Furthermore, the growth of S. typhimurium in the liver and spleen was more effectively restricted in Listeria-infected mice than in animals vaccinated with heat-killed S. typhimurium, even though the Listeria-infected animals possessed no demonstrable cross-reacting antibody to S. typhimurium. The lack of resistance in the mice vaccinated with heat-killed organisms could not be attributed to any deficiency of humoral factors, since the serum from these animals was as effective at promoting phagocytosis and killing by macrophages as serum from actively infected (and demonstrably resistant) mice. Conversely, Salmonella-infected mice were totally resistant to intravenous challenge with L. monocytogenes. The level of resistance in individual animals was related to the numbers of residual Salmonellae remaining in the tissues; mice with heavier residual infections being the more resistant. Specific antiserum from mice vaccinated with heat-killed S. typhimurium was found to be significantly protective only when the intraperitoneal route of challenge was employed. The foregoing studies have been interpreted to mean that enhancement of the microbicidal ability of macrophages is the mechanism of major importance in acquired resistance to S. typhimurium infection in mice.